Daniel Roxbury

Molecular-basis of single-walled carbon nanotube recognition by single-stranded DNA.

Hybrids of biological molecules and single-walled carbon nanotubes (SWCNT) have proven useful for SWCNT sorting and are enabling several biomedical applications in sensing, imaging, and drug delivery. In the DNA-SWCNT system, certain short (10-20mer) sequences of single-stranded DNA recognize specific SWCNT, allowing the latter to be sorted from a chirality diverse mixture. (1) However, little is known about the DNA secondary structures that underlie their recognition of SWCNTs. Using replica exchange molecular dynamics (REMD) of multiple strands on a single SWCNT, we report that DNA forms ordered structures on SWCNTs that are strongly DNA sequence and SWCNT dependent. DNA sequence (TAT)(4) on its recognition partner, the (6,5) SWCNT, (1) forms an ordered right-handed helically wrapped barrel, stabilized by intrastrand, self-stitching hydrogen bonds and interstrand hydrogen bonding. The same sequence on the larger diameter (8,7)-SWCNT forms a different and less-stable structure, demonstrating SWCNT selectivity. In contrast, homopolymer (T)(12), with weaker tendency for intrastrand hydrogen bonding, forms a distinctly left-handed wrap on the (6,5)-SWCNT, demonstrating DNA sequence specificity. Experimental measurements show that (TAT)(4) selectively disperses smaller diameter SWCNTs more efficiently than (T)(12), establishing a relationship between recognition motifs and binding strength. The developing understanding of DNA secondary structure on nanomaterials can shed light on a number of issues involving hybrids of nanomaterials and biological molecules, including nanomedicine, health-effects of nanomaterials, and nanomaterial processing.

Recognition ability of DNA for carbon nanotubes correlates with their binding affinity.

The ability to sort mixtures of carbon nanotubes (CNTs) based on chirality has recently been demonstrated using special short DNA sequences that recognize certain matching CNTs of specific chirality. In this work, we report on a study of the relationship between recognition sequences and the strength of their binding to the recognized CNT. We have chosen the (6,5) CNT and its corresponding DNA recognition sequences for investigation in this study. Binding strength is quantified by studying the kinetics of DNA replacement by a surfactant, which is monitored by following shifts in the absorption spectrum. We find that recognition ability correlates strongly with binding strength thus measured; addition or subtraction of just one base from the recognition sequence can enhance the kinetics of DNA displacement some 20-fold. The surfactant displaces DNA in two steps: a rapid first stage lasting less than a few seconds, followed by progressive removal lasting tens of minutes. The kinetics of the second stage is analyzed to extract activation energies. Fluorescence studies support the finding that the DNA sequence that recognizes the (6,5)-CNT forms a more stable hybrid than its close relatives.

DNA conjugated SWCNTs enter endothelial cells via Rac1 mediated macropinocytosis

Several applications of single-walled carbon nanotubes (SWCNT) as nanovectors in biological systems have been reported, and several molecular pathways of cellular entry have been proposed. We employed transmission electron microscopy, confocal fluorescent microscopy, and UV-vis spectroscopic analysis to confirm the internalization of DNA-SWCNT in human umbilical vein endothelial cells. Additionally, by using pharmacological inhibitors as well as genetic approaches, we have found that SWCNT is endocytosed through Rac1- GTPase mediated macropinocytosis in normal endothelial cells.

Hyperspectral Microscopy of Near-Infrared Fluorescence Enables 17-Chirality Carbon Nanotube Imaging.

The intrinsic near-infrared photoluminescence (fluorescence) of single-walled carbon nanotubes exhibits unique photostability, narrow bandwidth, penetration through biological media, environmental sensitivity, and both chromatic variety and range. Biomedical applications exploiting this large family of fluorophores will require the spectral and spatial resolution of individual (n,m) nanotube species’ fluorescence and its modulation within live cells and tissues, which is not possible with current microscopy methods. We present a wide-field hyperspectral approach to spatially delineate and spectroscopically measure single nanotube fluorescence in living systems. This approach resolved up to 17 distinct (n,m) species (chiralities) with single nanotube spatial resolution in live mammalian cells, murine tissues ex vivo, and zebrafish endothelium in vivo. We anticipate that this approach will facilitate multiplexed nanotube imaging in biomedical applications while enabling deep-tissue optical penetration, and single-molecule resolution in vivo.

Structural characteristics of oligomeric DNA strands adsorbed onto single-walled carbon nanotubes.

The single-stranded DNA to single-walled carbon nanotube (SWCNT) hybrid continues to attract significant interest as an exemplary biological molecule-nanomaterial conjugate. In addition to their many biomedical uses, such as in vivo sensing and delivery of molecular cargo, DNA-SWCNT hybrids enable the sorting of SWCNTs according to their chirality. Current experimental methods have fallen short of identifying the actual structural ensemble of DNA adsorbed onto SWCNTs that enables and controls several of these phenomena. Molecular dynamics (MD) simulation has been a useful tool for studying the structure of these hybrid molecules. In recent studies, using replica exchange MD (REMD) simulation we have shown that novel secondary structures emerge and that these structures are DNA-sequence and SWCNT-type dependent. Here, we use REMD to investigate in detail the structural characteristics of two DNA-SWCNT recognition pairs: (TAT)(4)-(6,5)-SWCNT, i.e., DNA sequence TATTATTATTAT bound to the (6,5) chirality SWCNT, and (CCG)(2)CC-(8,7)-SWCNT as well as off-recognition pairs (TAT)(4)-(8,7)-SWCNT and (CCG)(2)CC-(6,5)-SWCNT. From a structural clustering analysis, dominant equilibrium structures are identified and show a right-handed self-stitched motif for (TAT)(4)-(6,5) in contrast to a left-handed β-barrel for (CCG)(2)CC-(8,7). Additionally, characteristics such as DNA end-to-end distance, solvent accessible SWCNT surface area, DNA hydrogen bonding between bases, and DNA dihedral distributions have been probed in detail as a function of the number of DNA strands adsorbed onto the nanotube. We find that the DNA structures adsorbed onto a nanotube are also stabilized by significant numbers of non-Watson-Crick hydrogen bonds (intrastrand and interstrand) in addition to π-π stacking between DNA bases and nanotube surface and Watson-Crick pairs. Finally, we provide a summary of DNA structures observed for various DNA-SWCNT hybrids as a preliminary set of motifs that may be involved in the functional role of these hybrids.

Cell Membrane Proteins Modulate the Carbon Nanotube Optical Bandgap via Surface Charge Accumulation.

Cell adhesion is a protein-mediated process intrinsic to most living organisms. Dysfunction in cell adhesion processes is implicated in various diseases, including thrombosis and metastatic cancers. Using an approach to resolve spectral features from cell membrane-associated photoluminescent single-walled carbon nanotubes, we found that nanotube optical bandgaps respond to the electrostatic potential of the cell surface, which corresponds to cell adhesion properties. We studied the carbon nanotube emission energy response to solution ionic potentials, which suggests sensitivity to local charge accumulation. We conclude that nanotubes respond to cell surface electrostatic potentials that are mediated by membrane proteins, which vary significantly across cell types. These findings portend the optical measurement of surface electrostatic potentials for biophysical measurements and biomedical applications.

Helical polycarbodiimide cloaking of carbon nanotubes enables inter-nanotube exciton energy transfer modulation.

The use of single-walled carbon nanotubes (SWCNTs) as near-infrared optical probes and sensors require the ability to simultaneously modulate nanotube fluorescence and functionally derivatize the nanotube surface using noncovalent methods. We synthesized a small library of polycarbodiimides to noncovalently encapsulate SWCNTs with a diverse set of functional coatings, enabling their suspension in aqueous solution. These polymers, known to adopt helical conformations, exhibited ordered surface coverage on the nanotubes and allowed systematic modulation of nanotube optical properties, producing up to 12-fold differences in photoluminescence efficiency. Polymer cloaking of the fluorescent nanotubes facilitated the first instance of controllable and reversible internanotube exciton energy transfer, allowing kinetic measurements of dynamic self-assembly and disassembly.

A Carbon Nanotube Optical Sensor Reports Nuclear Entry via a Noncanonical Pathway

Single-walled carbon nanotubes are of interest in biomedicine for imaging and molecular sensing applications and as shuttles for various cargos such as chemotherapeutic drugs, peptides, proteins, and oligonucleotides. Carbon nanotube surface chemistry can be modulated for subcellular targeting while preserving photoluminescence for label-free visualization in complex biological environments, making them attractive materials for such studies. The cell nucleus is a potential target for many pathologies including cancer and infectious diseases. Understanding mechanisms of nanomaterial delivery to the nucleus may facilitate diagnostics, drug development, and gene-editing tools. Currently, there are no systematic studies to understand how these nanomaterials gain access to the nucleus. Herein, we developed a carbon nanotube based hybrid material that elucidate a distinct mechanism of nuclear translocation of a nanomaterial in cultured cells. We developed a nuclear-targeted probe via cloaking photoluminescent single-walled carbon nanotubes in a guanidinium-functionalized helical polycarbodiimide. We found that the nuclear entry of the nanotubes was mediated by the import receptor importin β without the aid of importin α and not by the more common importin α/β pathway. Additionally, the nanotube photoluminescence exhibited distinct red-shifting upon entry to the nucleus, potentially functioning as a reporter of the importin β-mediated nuclear transport process. This work delineates a noncanonical mechanism for nanomaterial delivery to the nucleus and provides a reporter for the study of nucleus-related pathologies.

A carbon nanotube reporter of microRNA hybridization events in vivo

MicroRNAs and other small oligonucleotides in biofluids are promising disease biomarkers, yet conventional assays require complex processing steps that are unsuitable for point-of-care testing or for implantable or wearable sensors. Single-walled carbon nanotubes are an ideal material for implantable sensors, owing to their emission in the near-infrared spectral region, photostability and exquisite sensitivity. Here, we report an engineered carbon-nanotube-based sensor capable of real-time optical quantification of hybridization events of microRNA and other oligonucleotides. The mechanism of the sensor arises from competitive effects between displacement of both oligonucleotide charge groups and water from the nanotube surface, which result in a solvatochromism-like response. The sensor, which allows for detection via single-molecule sensor elements and for multiplexing by using multiple nanotube chiralities, can monitor toehold-based strand-displacement events, which reverse the sensor response and regenerate the sensor complex. We also show that the sensor functions in whole urine and serum, and can non-invasively measure DNA and microRNA after implantation in live mice.

Photoluminescent carbon nanotubes interrogate the permeability of multicellular tumor spheroids

Nanomaterials have been extensively investigated for cancer drug delivery and imaging applications. Nanoparticles that show promise in two-dimensional cell culture systems often fail in more complex environments, possibly due to the lack of penetration in dense, three-dimensional structures. Multicellular tumor spheroids are an emerging model system to investigate interactions of nanoparticles with 3D in vitro cell culture environments. Using the intrinsic near-infrared emission of semiconducting carbon nanotubes to optically reconstruct their localization within a three-dimensional volume, we resolved the relative permeability of two different multicellular tumor spheroids. Nanotube photoluminescence revealed that nanotubes rapidly internalized into MCF-7 breast cancer cell-derived spheroids, whereas they exhibited little penetration into spheroids derived from SK-136, a cell line that we developed from murine liver cancer. Characterization of the spheroids by electron microscopy and immunohistochemistry revealed large differences in the extracellular matrix and interstitial spacing, which correlated directly with nanotube penetration. This platform portends a new approach to characterize the permeability of living multicellular environments.